Transformation of a Metal Chelate into a “Catch and Anchor” Inhibitor of Botulinum A Protease

Targeting the botulinum neurotoxin light chain (LC) metalloprotease using small-molecule metal chelate inhibitors is a promising approach to counter the effects of the lethal toxin. However, to overcome the pitfalls associated with simple reversible metal chelate inhibitors, it is crucial to investigate alternative scaffolds/strategies. In conjunction with Atomwise Inc., in silico and in vitro screenings were conducted, yielding a number of leads, including a novel 9-hydroxy-4H-pyrido [1,2-a]pyrimidin-4-one (PPO) scaffold. From this structure, an additional series of 43 derivatives were synthesized and tested, resulting in a lead candidate with a Ki of 150 nM in a BoNT/A LC enzyme assay and 17 µM in a motor neuron cell-based assay. These data combined with structure-activity relationship (SAR) analysis and docking led to a bifunctional design strategy, which we termed “catch and anchor” for the covalent inhibition of BoNT/A LC. Kinetic evaluation was conducted on structures prepared from this catch and anchor campaign, providing kinact/Ki values, and rationale for inhibition seen. Covalent modification was validated through additional assays, including an FRET endpoint assay, mass spectrometry, and exhaustive enzyme dialysis. The data presented support the PPO scaffold as a novel candidate for targeted covalent inhibition of BoNT/A LC.


Introduction
Botulinum neurotoxin A (BoNT/A) is the most toxic substance known, with an estimated i.v. lethal dose (LD 50 ) of 1-2 ng/kg in humans [1]. It is a 50 kDa protease secreted by the bacterium Clostridium botulinum and related strains found in soil. BoNTs have found extensive use in cosmetics and therapeutics for an increasing number of indications, including pain management, urinary incontinence, and muscle spasms [2]. While the use of BoNTs is generally considered safe, off-target effects and adverse events can result in anaphylaxis, muscle weakness, dysphagia, and iatrogenic botulism [3]. BoNTs A, B, and, less commonly, E and F are capable of intoxicating humans, causing botulism. In humans, botulism causes flaccid paralysis, which can result in life-threatening conditions due to asphyxiation of the diaphragm. There are a few hundred annual cases of botulism in the United States, mainly stemming from improper food preparation or wound infections by Clostridium botulinum [4,5]. Despite this rarity, the Center for Disease Control (CDC) has designated BoNTs as a Category A bioterrorism threat owing to their extreme toxicity, simplicity of preparation, ease of potential dissemination, and historical evidence of their attempted use [1,6,7]. A single bioterrorism event could easily overwhelm lean healthcare infrastructure, as ventilators and long-term hospitalization are required for treatment. The current standard of care for botulism is treatment with an equine-derived antibody, most effective if administered within 24 h of intoxication [8,9]. Because antibodies are incapable of sequestering and inhibiting BoNTs after they enter neuronal compartments, they become significantly less effective after this initial window has passed. In addition, there is a risk of adverse reactions such as anaphylaxis occurring. Patients are required to remain hospitalized for weeks to months, often on mechanical ventilation and parenteral nutrition, during the course of treatment [6,7,10].
Many small-molecule drugs are capable of penetrating cells and are thus a promising strategy for meeting this unmet need. The BoNT holotoxin is composed of a heavy chain (HC) translocation domain that mediates the internalization of the light chain (LC), a zinc-dependent metalloprotease, into motor neurons. Thus, non-peptidic small-molecule inhibitors of BoNT usually target the active site of BoNT LC and typically contain a metal binding group (MBG) [11]. However, no such inhibitors have progressed to clinical trials. The difficulty of development can be partially attributed to the disparity between the halflife of BoNT/A LC in neurons and the drug in the body-the former persists for months, while the latter is cleared over a time frame of hours to days [12,13]. In our search for a clinically viable inhibitor of BoNT/A LC, we pivoted from the design of conventional reversible inhibitors to what we term a "catch and anchor" strategy. The general concept involves the tethering of a covalent warhead to an active-site inhibitor scaffold. Moreover, this approach leverages the high affinity afforded by an MBG, the specificity and complete inhibition of an active-site inhibitor, and the longevity of the covalent adduct.
Despite its putative low reactivity, Cys165 remains an attractive modality for covalent modification due to its position near the active site [14]. Despite not being involved directly in the catalytic cycle or structural stability, Cys165 is an important residue, as its mutation is associated with a 50-fold decrease in catalytic activity [15]. It is also understood that the active site of BoNT/A LC tolerates a significant amount of plasticity while maintaining the same conformation of zinc and the catalytic residues; however, the catalytic cleft microenvironment can be altered depending on the inhibitor that is bound [16,17]. Moreover, hydroxamic acids, the most commonly used MBG for BoNT/A LC inhibition, face significant stability issues in the physiological environment [18,19]. Thus, by investigating new inhibitor scaffolds, we aim to address the drawbacks of our previous efforts, namely stability, cell permeability, and potency. To this end, we describe the discovery and development of a new class of BoNT/A LC inhibitors and their subsequent advancement to a targeted covalent inhibitor strategy.

Screening Campaign
In collaboration with Atomwise Inc., the search began with an in silico screen of inhibitors using the company's AtomNet ® model. The AtomNet ® model is the first deep neural network for structure-based drug discovery [20]. A single global AtomNet ® model was trained on a large number of protein structures and bioactivity data of small-molecule binders, as previously described [16]. The sglobal AtomNet ® model was then used to screen the eMolecules diverse library of 1 million molecules against the X-ray crystal structure of 2,4-dichlorocinnamyl hydroxamic acid bound to BoNT/A LC (PDB 2IMA) [16]. The 33 top-scoring hits were purchased from eMolecules and screened at a final concentration of 40 µM against BoNT/A LC in the SNAPtide FRET assay, a robust FRET assay that has led to the discovery of inhibitors with in vivo efficacy [21][22][23][24]. This screening effort yielded 9-hydroxy-4H-pyrido [1,2-a]pyrimidin-4-one (PPO)-based 1 ( Figure 1A) as a top hit. Next, a focused sub-library of 26 derivatives was subsequently ranked in silico with AtomNet and screened within the SNAPtide FRET assay ( Figure 1B). Four members (including 1) of the PPO sub-library inhibited BoNT/A LC activity by more than 90%. Compounds 2 and 3, both of which contained six-membered heterocycles, were found to be approximately seven-fold more potent than initial lead 1 ( Figure 1A). Based upon these results, 2 was selected for further optimization due to its inhibitory potency, ease of synthesis, and high potential for derivatization due to the secondary amine embedded within its structure. In passing, we note that the chiral centers found within the pyrrolidine ring of the equipotent

BoNT/A LC Inhibition & SAR
The PPO scaffold was further probed by syntheses of compounds 5-14 (Scheme 1) to determine the minimum pharmacophore. Analogous to the majority of non-peptidic small-molecule inhibitors of BoNT/A LC, PPO is a metal chelating scaffold that binds Zn 2+ with the oxygen of its hydroxyl group and the nitrogen in the pyridopyrimidinone heterocycle [8]. The exclusion of the hydroxyl group (10) resulted in the complete ablation of inhibitory activity, while masking the hydroxyl functionality with an ethyl group (11) had a similar effect, which is consistent with its known metal binding mechanism [8].  (1)(2)(3)(4). Values are reported as mean ± SD, n = 3. (B) Screening results of the PPO sub-library, tested at 40 µM in duplicate. Data were normalized to a DMSO control.

BoNT/A LC Inhibition & SAR
The PPO scaffold was further probed by syntheses of compounds 5-14 (Scheme 1) to determine the minimum pharmacophore. Analogous to the majority of non-peptidic small-molecule inhibitors of BoNT/A LC, PPO is a metal chelating scaffold that binds Zn 2+ with the oxygen of its hydroxyl group and the nitrogen in the pyridopyrimidinone heterocycle [8]. The exclusion of the hydroxyl group (10) resulted in the complete ablation of inhibitory activity, while masking the hydroxyl functionality with an ethyl group (11) had a similar effect, which is consistent with its known metal binding mechanism [8]. To examine the impact of the benzylamine substituent, the terminal methyl analogue (6) and the primary amine (13) were synthesized. The loss of phenyl groups resulted in a decrease in inhibitory potency for both compounds. This, in combination with the complete lack of inhibitory activity of intermediates, suggests that the benzyl To examine the impact of the benzylamine substituent, the terminal methyl analogue (6) and the primary amine (13) were synthesized. The loss of phenyl groups resulted in a decrease in inhibitory potency for both compounds. This, in combination with the complete lack of inhibitory activity of intermediates, suggests that the benzyl group interacts favorably with the active site and that the Zn-chelating group alone is not sufficient for binding. Additionally, based on the IC 50 values of 1-4, it is evident that a variety of heterocycles can be tolerated. We also posit that the nitrogen atom plays a role in potency, as replacing the secondary amine with a methylene group to granting 7 resulted in a drastic decrease in inhibitory activity. This reduction in inhibitory activity suggests that the secondary amine is not directly involved in metal chelation but likely engages in hydrogen bond interactions. Finally, quinolinol derivative 14 showed a 15-fold decrease in inhibition, implying that the PPO carbonyl may promote additional hydrogen bonding interactions.
As determined in past SAR campaigns, which also align with crystallographic analysis, aromatic groups tend to sit in the hydrophobic pocket, which contains aromatic residues amenable for π-π stacking [3,9,10,17]. Hence, the PPO-SAR study was focused on this region of the molecule. Compounds were synthesized from their corresponding benzylamines, benzyl alcohols, and N-containing heterocycles under the same conditions as those presented in Scheme 1A, and IC 50 values were obtained and summarized in Table 1. Based on the trends observed for benzylamine derivatives 2-29, some general conclusions could be drawn. Irrespective of their electronic properties, 3 -substituted molecules were around 3-fold more potent than their 4 -substituted counterparts, which suggests that activity depends less on electrostatic interactions but more on the positioning that the substituent occupies in the active site. This effect can be seen by comparing methyl ester structural isomers 24 and 25; 3 -substituted methyl ester 24 is among the most potent of benzylamine derivatives, with an IC 50 of 0.41 ± 0.08 µM. Conversely, the 4 -substituted analogue 25 is poorly positioned, so that inhibition is abolished (IC 50 > 64 µM). Interestingly, the addition of a phenyl group to the benzyl ring at the 4 -position (27) significantly increased inhibitory potency compared to 2, while substitution at the 3 -position proved slightly less potent (26). The K i of compound 27 was calculated to be 150 nM based on the Cheng-Prusoff equation for a competitive inhibitor, where substrate K m = [S] [25].
Past studies have reported that the BoNT/A LC active site can accommodate very large substituents and that inhibitor binding is then accompanied by a significant rearrangement of hydrophobic side chains in the active site, with retention of the geometry of the catalytic triad. This can be seen in the structural differences between X-ray crystal structures bound with the small 2,4-dichlorocinnamyl hydroxamic acid and a large adamantane hydroxamic acid inhibitor (PDB 2IMA and 4HEV, respectively) [3,9]. It is possible that the active site of BoNT/A LC accommodates the large PPO inhibitors in a similar manner.
Due to the protonation of the secondary amine at physiological pH, cell permeability could be compromised and therefore impact efficacy. Thus, a series of benzyl ether derivatives (30-44, Table 1) were synthesized to replace the amine, which afforded a series of 10-fold less potent analogues compared to the benzylamine series. The two main consequences of replacing the amine with an oxygen atom are the loss of an H-bond donor and the change in bond geometry. It is possible that both contribute to the lower inhibitory potency of this series. In addition to benzylamine and benzyl ethers, a limited series of isoindoline and indoline derivatives were prepared and examined. The isoindolines can be thought of as a constrained benzyl group and were synthesized to offer some insight into benzyl group positioning. Interestingly, we found that isoindoline 42 was equipotent to 2. This lack of improvement in potency suggests that this is not likely the optimal orientation of the benzyl group, while any benefit stemming from the reduction in entropy afforded by the restrained benzyl group of 42 could be counteracted by slightly less favorable binding. large substituents and that inhibitor binding is then accompanied by a significant rearrangement of hydrophobic side chains in the active site, with retention of the geometry of the catalytic triad. This can be seen in the structural differences between Xray crystal structures bound with the small 2,4-dichlorocinnamyl hydroxamic acid and a large adamantane hydroxamic acid inhibitor (PDB 2IMA and 4HEV, respectively) [3,9]. It is possible that the active site of BoNT/A LC accommodates the large PPO inhibitors in a similar manner. To validate the inhibition of BoNT/A LC by the PPO compounds detailed, vide supra, a number of the most potent derivatives were evaluated in a cell assay that quantifies intact and cleaved SNAP-25 in human-induced pluripotent stem cell (hiPSC)-derived motor neurons. This cell assay has been used extensively to evaluate BoNT/A inhibitors, offering further insight into the external factors that affect inhibitor potency. The compounds presented in Figure 2 were selected based on potency and diversity. Surprisingly, neither 2 nor 18 inhibited the cleavage of SNAP-25 in the cellular assay despite their excellent inhibitory activity in the enzyme assay. We posit that the lack of inhibitory activity could be related to poor cell permeability stemming from the polarity of the secondary nitrogen, which would exist primarily in the protonated state at physiological pH. Notably, benzyl ether derivative 32, which is nearly 10-fold less potent than 2 in the enzyme assay (Table 1), showed nearly complete protection of SNAP-25 at 100 µM. Similarly, isoindolines 47 and 54 both protect against SNAP-25 cleavage at 100 µM, although to a lesser degree. Interestingly, although 35 is also a benzyl ether derivative, it did not have any apparent effect on SNAP-25 cleavage. In sum, of the compounds tested, 27 was the most potent, with an IC 50 of 13 µM (12-16 µM, 95% CI).  Figure S1. The upper bands represent intact SNAP-25, and the lower bands represent cleaved SNAP-25. The positive control group was exposed to BoNT/A, and the negative control group was not. Both control groups were treated with cell media containing 1% DMSO.

Docking-Based Covalent Linker Design
The promising inhibitory potency in both the in vitro enzyme and cell assays prompted the selection of 27 for lead development as a catch and anchor inhibitor. An Xray crystal structure of BoNT/A LC co-crystallized with an adamantane inhibitor was used for docking ( Figure 3A), as the larger hydrophobic pocket generated from this structure could accommodate the bulkier structural sizes currently under investigation [26]. Based on the lowest energy poses, it appeared that the heteroatom can form a hydrogen bond with Tyr366, echoing the experimental data obtained. The lowestscoring compounds were molecules that did not interact with the hydrophobic pocket, and correlated with poor IC50 values.  Figure S1. The upper bands represent intact SNAP-25, and the lower bands represent cleaved SNAP-25. The positive control group was exposed to BoNT/A, and the negative control group was not. Both control groups were treated with cell media containing 1% DMSO.

Docking-Based Covalent Linker Design
The promising inhibitory potency in both the in vitro enzyme and cell assays prompted the selection of 27 for lead development as a catch and anchor inhibitor. An X-ray crystal structure of BoNT/A LC co-crystallized with an adamantane inhibitor was used for docking ( Figure 3A), as the larger hydrophobic pocket generated from this structure could accommodate the bulkier structural sizes currently under investigation [26]. Based on the lowest energy poses, it appeared that the heteroatom can form a hydrogen bond with Tyr366, echoing the experimental data obtained. The lowest-scoring compounds were molecules that did not interact with the hydrophobic pocket, and correlated with poor IC 50 values.
As detailed, vide supra, this strategy combines an active site binding scaffold with a pendent Cys-reactive warhead to achieve targeted covalent inhibition of BoNT/A LC [15,22]. Moreover, the success of the catch and anchor strategy is dependent on the proximity of the reactive warhead to the cysteine residue of interest, while simultaneously engaging the metal center. Based on the lowest energy position of the PPO derivatives shown in Figure 3A, it was determined that extension from position 8 would provide the optimal vector for a reactive warhead to engage with Cys165, with an approximate distance of 6-7 Å between C8 and Cys165. Using docking as our guide, a preliminary molecule was sought with a thiol-reactive methanethiosulfonate (MTS) warhead, and a linker length predicted to be n = 4, approximately 6.6 Å ( Figure 3B). Ease of attachment and high thiol reactivity make As detailed, vide supra, this strategy combines an active site binding scaffold with a pendent Cys-reactive warhead to achieve targeted covalent inhibition of BoNT/A LC [15,22]. Moreover, the success of the catch and anchor strategy is dependent on the Instead, intermediate 57 was O-methylated. Here, a felicitous trans-halogenation occurred during the methylation reaction, forming 58 in the presence of excess alkylating reagent. This caused a dramatic acceleration of the subsequent SN2 reaction compared to the corresponding chloride intermediate, while Boc protection yielded the fully protected 8-iodofunctionalized 59. As an added advantage, the presence of the Boc and methoxy protecting groups drastically eased purification. Scaffolds with various linker lengths (60-64) were then accessed by Pd-catalyzed Sonogashira coupling between 59 and the corresponding alkynyl alcohols. Hydrogenation (65-69) and subsequent bromination yielded protected alkyl bromide compounds (70-74). Although Sonogashira coupling led to a high yield, the resulting compounds could not be hydrogenated. Double deprotection of 70-74 was achieved by treatment with BBr3 in anhydrous DCM in quantitative yield to furnish the penultimate alkyl bromide intermediates (75-79), which were functionalized by substitution with sodium methanethiosulfonate to yield catch and anchor compounds 80-84.

Catch and Anchor Inhibition
This new series of catch and anchor compounds was first screened in the continuous SNAPtide enzyme assay, presenting time-dependent inhibition ( Figure S2). MTS molecules have been shown to covalently inhibit BoNT/A LC [14,22], but it was nevertheless necessary to validate irreversible inhibition. PPO 2 and parent 27 were included as reversible controls in all experiments. To assess long-term time-dependent inhibition, an endpoint assay was performed with 81, which was found to inhibit BoNT/A LC in a time-dependent manner ( Figure 4A). Scaffolds with various linker lengths (60-64) were then accessed by Pd-catalyzed Sonogashira coupling between 59 and the corresponding alkynyl alcohols. Hydrogenation (65-69) and subsequent bromination yielded protected alkyl bromide compounds (70-74). Although Sonogashira coupling led to a high yield, the resulting compounds could not be hydrogenated. Double deprotection of 70-74 was achieved by treatment with BBr 3 in anhydrous DCM in quantitative yield to furnish the penultimate alkyl bromide intermediates (75-79), which were functionalized by substitution with sodium methanethiosulfonate to yield catch and anchor compounds 80-84.

Catch and Anchor Inhibition
This new series of catch and anchor compounds was first screened in the continuous SNAPtide enzyme assay, presenting time-dependent inhibition ( Figure S2). MTS molecules have been shown to covalently inhibit BoNT/A LC [14,22], but it was nevertheless necessary to validate irreversible inhibition. PPO 2 and parent 27 were included as reversible controls in all experiments. To assess long-term time-dependent inhibition, an endpoint assay was performed with 81, which was found to inhibit BoNT/A LC in a time-dependent manner ( Figure 4A).
In order to rule out slow-binding kinetics as a cause of time-dependent inhibition, a dialysis experiment was conducted. BoNT/A LC was incubated with 5 µM of 81 for 30 min, and its enzyme activity was measured before and after exhaustive dialysis ( Figure 4B). Enzyme incubated with 81 only showed~60% of enzyme activity compared to the DMSO control, which was not recovered following dialysis. Since the enzyme samples were exhaustively dialyzed over 18 h, it can be concluded that the observed time-dependent inhibition is not a result of slow binding, but rather of covalent modification. Finally, covalent adduct formation was probed using HRMS. As in previous reports, the BoNT/A LC C165S variant was included as a method to differentiate the two cysteines present on BoNT/A LC [14,22]. As expected, only BoNT/A LC WT that had been incubated with either 81 or 82 showed an increase in mass matching two covalent adducts, which corresponded to adduct formation at both the active-site proximal Cys165 and distal Cys134 of the WT. Neither of the reversible compounds 2 and 27 showed any change in mass. When the C165S variant was incubated with either 81 or 82, a mass increase corresponding to the addition of one adduct was observed. Because Ser165 of the C165S variant is unreactive to methanethiosulfonate, it can be concluded that the compounds indeed label BoNT/A LC WT at Cys165. In order to rule out slow-binding kinetics as a cause of time-dependent inhibition, a dialysis experiment was conducted. BoNT/A LC was incubated with 5 µ M of 81 for 30 min, and its enzyme activity was measured before and after exhaustive dialysis ( Figure  4B). Enzyme incubated with 81 only showed ~60% of enzyme activity compared to the DMSO control, which was not recovered following dialysis. Since the enzyme samples were exhaustively dialyzed over 18 h, it can be concluded that the observed timedependent inhibition is not a result of slow binding, but rather of covalent modification. Finally, covalent adduct formation was probed using HRMS. As in previous reports, the BoNT/A LC C165S variant was included as a method to differentiate the two cysteines present on BoNT/A LC [14,22]. As expected, only BoNT/A LC WT that had been incubated with either 81 or 82 showed an increase in mass matching two covalent adducts, which corresponded to adduct formation at both the active-site proximal Cys165 and distal Cys134 of the WT. Neither of the reversible compounds 2 and 27 showed any change in mass. When the C165S variant was incubated with either 81 or 82, a mass increase corresponding to the addition of one adduct was observed. Because Ser165 of the C165S variant is unreactive to methanethiosulfonate, it can be concluded that the compounds indeed label BoNT/A LC WT at Cys165. Following the validation of covalent irreversible inhibition, the k inact /K i values of these compounds were obtained using the previously described continuous SNAPtide assay and are reported in (Table 2) [22]. A clear trend could be seen between linker length and k inact /K i , which indicated that a linker of four carbons (81) would be ideal. This compound had a k inact /K i value that is four-fold higher than the analogous proof-ofconcept compound that utilizes the 2,4-dichlorocinammic hydroxamate (DCHA) scaffold (k inact /K i = 514 ± 17 M −1 s −1 ) [14]. The effect of linker length and warhead proximity to Cys165 is extremely informative within this series of compounds; there is more than a two-fold difference between compounds 81 and 82, and seven-fold between 81 and 80, with only one carbon difference in linker length. It is likely that the difference in potency between 81 and 80 versus 81 and 82 is due to the linker length being too constrained; thus, the needed bridge between zinc ion and Cys165 is ultimately compromised. Conversely, the channel that the linker resides in may accommodate additional length. To compare linker lengths between the PPO series and the previously published DCHA series, the energy-minimized conformation of compound 81 was obtained in Chem3D. The length from the chelating group to the reactive moiety was measured to be approximately 6.9 Å, which is slightly shorter than the 7.8 Å measured in the X-ray crystal structure of the optimal chain length obtained in the proof-of-concept study. Furthermore, the distance between the reactive cysteine and C8 was measured to be 6.3 Å, close to the predicted distance of 6.1 Å obtained from docking ( Figure 3A). Although Chem3D provides the minimum energy in the absence of the enzyme structure, the similarity between actual, predicted, and optimal linker lengths underlines the importance of obtaining structural information. An overall comparison of the inhibitory constants shows that k inact remained in the same range and that the difference in potency stemmed from differences in K i . As we have noted, vide supra, this series of inhibitors are functionalized with an extremely reactive MTS group, which, unlike less reactive warheads, does not necessarily require active site targeting to be effective. In fact, as exemplified by the mass spectrometry experiments ( Figure 4C), the MTS group react with any available cysteine. Thus, we posit that the potency of these compounds is governed by their affinity for the enzyme (K i ) active site rather than their rate of enzyme inactivation (k inact ). This improvement and emphasis on K i will allow the incorporation of less reactive warheads without compromising on potency; such efforts are in progress to test this hypothesis. Table 2. Inhibition kinetics of catch and anchor PPO compounds. Values represent mean ± SD, n = 3. All biochemical analyses and cell assays were performed as previously reported by Lin et al. [22] and Turner, Nielsen et al. [14] with the following modifications.

Screening
Initial compound screening was performed at room temperature with SNAPtide substrate #521 (List Labs, Campbell, CA, USA) at a compound concentration of 40 µM.

Endpoint Assay
In total, 500 nM BoNT/A LC was incubated with 5 µM of each compound for 30 min at 37 • C. At the indicated timepoints, 2 µL aliquots were quenched in 48 µL of SNAPtide #523 substrate (25-fold dilution) to give a SNAPtide assay final concentration of 20 nM BoNT/A LC and 0.2 µM compound.

Dialysis Assay
Inhibitors were incubated at a concentration of 5 µM 500 nM BoNT/A LC for 30 min at 37 • C in assay buffer (40 mM HEPES, 0.01% Triton X-100, pH 7.4), 1% DMSO. Control samples containing no inhibitors were also prepared. Enzyme activity was evaluated before and after dialysis in the SNAPtide assay. Data analysis proceeded as previously described.

Hi-resolution Mass Spectrometry of Covalent Adducts
Inhibitors were incubated with BoNT/A LC WT for 30 min at 37 • C at a concentration of 100 µM inhibitor, 40 µM enzyme in assay buffer (40 mM HEPES, 0.01% Triton X-100, pH 7.4), and 1% DMSO. Control samples containing only enzymes in assay buffer were also analyzed. Putative masses of the covalent adducts were determined using ChemDraw 19. HRMS analysis of proteins was carried out as described previously. Expected protein masses were determined using the published construct sequences and ProtParam https: //web.expasy.org/protparam/ (accessed on 18 June 2020), and subtracting the first five fragmented residues (MGSSH).

hiPSC Motor Neuron Cell Assay
BoNT/A cell activity assays were performed as previously reported. Human-induced pluripotent stem cell (hiPSC)-derived GABA neurons and culture medium (Fujifilm Cellular Dynamics International (Madison, WI, USA) were cultured in 0.01% poly-L-ornithine (Sigma Aldrich, Burlington, MA, USA) and 8.3 µg/cm 2 matrigel, growth factor reduced, (BD Biosciences) coated 96-well TPP plates (Midsci) for 12 days prior to the assay. 200 LD50 Units of BoNT/A1 (150 kDa purified as described previously,15 specific activity (1.7 × 108 U/mg) was added to the cells in 50 µL stimulation medium (modified neurobasal containing 2.2 mM CaCl 2 and 56 mM KCl and supplemented with 1 × B27 and 1 × GlutaMAX (100×-stocks, Life Technologies, Carlsbad, CA, USA), and the cells were incubated at 37 • C in a humidified 5% CO 2 atmosphere for 7.5 min. Toxin was removed, cells were washed twice in 300 µL of culture medium, and further incubated in fresh culture medium at 37 • C in a humidified 5% CO 2 atmosphere. At 30 min post first toxin addition, the inhibitors were added at the indicated concentrations and with a final DMSO concentration of 1%. Positive control (+C) was toxin without inhibitor in culture media, and negative control (-C) was culture media, both with 1% DMSO added. Cells were incubated for 7.5 h post toxin addition at 37 • C, 5% CO 2 to allow for SNAP-25 cleavage. Inhibitor mixtures were then aspirated, and cells lysed in 50 µL of 1 × LDS lysis buffer (Invitrogen, Waltham, MA, USA). Cell lysates were analyzed by Western blot using a monoclonal anti-SNAP-25 antibody (Synaptic Systems, Göttingen, Germany; cat. #111011) as described previously, and bands were visualized using Phosphaglo chemiluminescent reagent (KPL) on an Azure C600 imaging system equipped with a CCD camera (Azure Biosystems). Assays were performed in triplicate.

Molecular Modeling
Molecular modeling was performed with modules from the Schrödinger Small Molecule Drug Discovery Suite (Maestro), release 2018-3, using the OPLS3 force field for parameterization. The X-ray co-crystal structure of BoNT/LC (PDB 4HEV) was imported from the protein data bank and prepared using the Protein Preparation Wizard using default settings. Compounds were prepared in LigPrep, and docked using the XP Glide docking module (using metal-binding constraint) [28]. The lowest energy binding mode of compound 22 is visualized in Figure 3, and the image was generated using the PyMol Molecular Graphics System (version 2.3.4., Schrödinger, LLC., New York, NY, USA).

General Procedures and Instrumentation
Reactions were carried out under atmospheric conditions, unless otherwise stated. All reagents were obtained from Sigma, Combi-blocks, or Fisher and used without further purification. Anhydrous solvents were distilled and stored over 4 Å molecular sieves. The reactions were monitored using thin-layer chromatography (TLC) or high-performance liquid chromatography-MS (HPLC-MS). TLC was performed using Merck precoated analytical plates (0.25 mM thick silica gel 60 F254). HPLC-MS analysis was performed on an Agilent 1260 Infinity II instrument coupled to a single quadrupole InfinityLab LC/MSD instrument running a gradient of eluent I (0.1% formic acid in H 2 O) and eluent II (0.1% formic acid in MeCN) rising linearly from 0 to 95% of II during t = 0.00-6.00 min and then with eluent II from t = 6.00-10.0 min, at a flow rate of 0.5 mL/min on a Zorbax 300SB-C8 column at 35 • C. Flash automated column chromatography (ACC) was performed using a CombiFlash Rf+ Lumen (Teledyne Isco) purification system with flash silica RediSep Rf columns for normal phase (NP), amine-functionalized silica RediSep Rf columns for normal phase, or RediSep Rf Gold C18 HP columns for reversed phase (RP). The purity of all tested compounds is >95%, as determined by HPLC-MS.

2-((Benzylamino)methyl)quinolin-8-ol (14)
In an oven-dried flask under argon, 8-hydroxylquinoline-2-carboxaldehyde (173 mg, 1.0 mmol, 1.0 equiv.) and benzylamine (120 µL, 1.2 mmol, 1.2 equiv.) were dissolved in 5 mL anhydrous dichloroethane and cooled to 0 • C in an ice bath. Sodium triacetoxyborohydride (317 mg, 1.5 mmol, 1.0 equiv.) was added in three portions, flushing the vessel with argon after each addition. The reaction was stirred for 5 min in the ice bath before the bath was removed and the reaction allowed to warm to room temperature. After 24 h, 10 mL H 2 O was added slowly to quench the reaction. The reaction mixture was extracted with 20 mL DCM, and the organic extract was dried over Na 2 SO4 and concentrated. The crude product was purified using normal-phase ACC on amine-functionalized silica (0-50% EtOAc in hexanes) to yield 222 mg (0.84 mmol, 84.1%) yellow solid as the title compound. 1